PROJECT SUMMARY Viruses in the Coronaviridae family (CoVs) have emerged as zoonoses with pandemic potential twice in the 21st century, causing severe human disease. Middle East respiratory syndrome (MERS)-CoV continues to cause new cases of lethal respiratory infections with 35% mortality. Further, severe acute respiratory syndrome (SARS)-like bat CoVs currently circulating are capable of infecting human cells, establishing the risk for future emergence of zoonotic CoVs. There are no approved vaccines or antivirals for any human or zoonotic CoV, emphasizing the importance of identifying vulnerable and broadly conserved CoV targets for therapeutic intervention and vaccine development. Most RNA viruses generate genetic diversity required for interspecies movement and adaptation via error-prone RNA-dependent RNA polymerases (RdRps) that lack proofreading. In contrast, all CoVs encode a 3'-to-5' exoribonuclease (ExoN) in nonstructural protein 14 (nsp14-ExoN) that is a key driver of CoV evolution and adaptation via RNA-dependent RNA proofreading. During the four years of funding for this program, we have shown that CoV nsp14-ExoN mediates high-fidelity replication and that CoVs lacking ExoN activity (ExoN(-)) are less fit during infection in cell culture, more sensitive to RNA mutagens, and attenuated in a murine model of SARS-CoV infection. Our findings suggest that divergent ?-CoVs - MERS-CoV, SARS-CoV, and murine hepatitis virus (MHV) - have differential requirements for ExoN to sustain viability and overall fitness. Finally, ExoN may play important and previously unpredicted functions in CoV resistance to host innate immune surveillance. Thus, our published and preliminary studies support the scientific premise that nsp14-ExoN is a master regulator of CoV fitness, evolution, and pathogenesis via functions in viral replication, fidelity, and evasion of host innate immune responses. Specific aims of this proposal will define: 1) Sequence and structural determinants of nsp14-ExoN-mediated functions in CoV replication, fidelity, and interferon sensitivity; 2) Adaptations in nsp14, nsp12-RdRp, and elsewhere in the CoV replicase that compensate for loss of ExoN-mediated fidelity; and 3) Mechanisms of ExoN regulation of the innate antiviral immune response in vitro and in vivo. The availability of a high-resolution structure of nsp14; facile reverse genetics systems for MHV, SARS-CoV, and MERS-CoV; and robust, relevant animal models for SARS-CoV and MERS-CoV will allow us to address these questions, resulting in a comprehensive understanding of ExoN roles and mechanisms in CoV replication, adaptation, and pathogenesis. These studies will catalyze approaches targeting ExoN as a basis for stably attenuated CoV vaccines and novel antiviral drugs.